Nonspecific Protein Kinase Inhibitor Research Articles

Hypertonicity during a rapid rise in D-glucose mediates first-phase insulin secretion.

Biphasic insulin secretion is an intrinsic characteristic of the pancreatic islet and has clinical relevance due to the loss of first-phase in patients with Type 2 diabetes. As it has long been shown that first-phase insulin secretion only occurs in response to rapid changes in glucose, we tested the hypothesis that islet response to an increase in glucose is a combination of metabolism plus an osmotic effect where hypertonicity is driving first-phase insulin secretion. Experiments were performed using perifusion analysis of rat, mouse, and human islets. Insulin secretion rate (ISR) and other parameters associated with its regulation were measured in response to combinations of D-glucose and membrane-impermeable carbohydrates (L-glucose or mannitol) designed to dissect the effect of hypertonicity from that of glucose metabolism. Remarkably, the appearance of first-phase responses was wholly dependent on changes in tonicity: no first-phase in NAD(P)H, cytosolic calcium, cAMP secretion rate (cAMP SR), or ISR was observed when increased D-glucose concentration was counterbalanced by decreases in membrane-impermeable carbohydrates. When D-glucose was greater than 8 mM, rapid increases in L-glucose without any change in D-glucose resulted in first-phase responses in all measured parameters that were kinetically similar to D-glucose. First-phase ISR was completely abolished by H89 (a non-specific inhibitor of protein kinases) without affecting first-phase calcium response. Defining first-phase ISR as the difference between glucose-stimulated ISR with and without a change in hypertonicity, the peak of first-phase ISR occurred after second-phase ISR had reached steady state, consistent with the well-established glucose-dependency of mechanisms that potentiate glucose-stimulated ISR. The data collected in this study suggests a new model of glucose-stimulated biphasic ISR where first-phase ISR derives from (and after) a transitory amplification of second-phase ISR and driven by hypertonicity-induced rise in H89-inhibitable kinases likely driven by first-phase responses in cAMP, calcium, or a combination of both.

Post-Translational Regulation of a Bidomain Glycerol-3-Phosphate Dehydrogenase Catalyzing Glycerol Synthesis under Salinity Stress in Chlamydomonas reinhardtii

Core chlorophytes possess glycerol-3-phosphate dehydrogenases (GPDs) with an unusual bidomain structure, consisting of a glycerol-3-phosphate phosphatase (GPP) domain fused to canonical GPD domains. These plastid-localized enzymes have been implicated in stress responses, being required for the synthesis of glycerol under high salinity and triacylglycerols under nutrient deprivation. However, their regulation under varying environmental conditions is poorly understood. C. reinhardtii transgenic strains expressing constitutively bidomain GPD2 did not accumulate glycerol or triacylglycerols in the absence of any environmental stress. Although the glycerol contents of both wild type and transgenic strains increased significantly upon exposure to high salinity, cycloheximide, an inhibitor of cytoplasmic protein synthesis, abolished this response in the wild type. In contrast, GPD2 transgenic strains were still capable of glycerol accumulation when cultured in medium containing cycloheximide and NaCl. Thus, the pre-existing GPD2 protein appears to become activated for glycerol synthesis upon salt stress. Interestingly, staurosporine, a non-specific inhibitor of protein kinases, prevented this post-translational GPD2 protein activation. Structural modeling analyses suggested that substantial conformational rearrangements, possibly triggered by high salinity, may characterize an active GPD2 GPP domain. Understanding this mechanism(s) may provide insights into the rapid acclimation responses of microalgae to osmotic/salinity stress.

Protein Kinases Alter the Allosteric Modulation of the Serotonin Transporter In Vivo and In Vitro.

Studies using S- and R-enantiomers of the SSRI citalopram have shown that R-citalopram exerts an antagonistic effect on the efficacy of the antidepressant S-citalopram (escitalopram) through an interaction at an allosteric modulator site on the serotonin transporter (SERT). Here, we show that protein kinase signaling systems are involved in the allosteric modulation of the SERT invivo and invitro. We assessed the effects of nonspecific protein kinase inhibitor staurosporine in the action of escitalopram and/or R-citalopram using electrophysiological and behavioral assays in rats and cell surface SERT expression measures in serotoninergic cells. Acute administration of R-citalopram counteracted the escitalopram-induced suppression of the serotonin (5-HT) neuronal firing activity and increase of the head twitches number following L-5-hydroxytryptophan injection. Importantly, these counteracting effects of R-citalopram were abolished by prior systemic administration of staurosporine. Interestingly, the preventing effect of staurosporine on 5-HT neuronal firing activity was abolished by direct activation of protein kinase C with phorbol 12-myristate 13-acetate. Finally, invitro, quantification of the amount of cell surface-expressed SERT molecules revealed that R-citalopram prevented escitalopram-induced SERT internalization that was completely altered by staurosporine. Taken together, these results highlight for the first time an involvement of protein kinases in the allosteric modulation of SERT function.

PKA and phosphatases attached to the CaV1.2 channel regulate channel activity in cell-free patches

Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca(2+) channels (Ca(V)1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the Ca(V)1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of Ca(V)1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. Ca(V)1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the Ca(V)1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced Ca(V)1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the Ca(V)1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.

Anti-tumor activity of staurosporine in the tumor microenvironment of cervical cancer: An in vitro study.

The fundamental events for cancer progression and metastases include loss of cell adhesion, cell proliferation, anchorage-independent cell growth (evading anoikis), cell migration and cell invasion. All these events leading to cancer progression happen in a favorable nurturing tumor microenvironment. This study was designed to explore the anti-tumor activity of staurosporine (a nonspecific protein kinase inhibitor) in the tumor microenvironment of cervical cancer. The anti-tumor activity of staurosporine was investigated by cell adhesion assay, colony formation assay, apoptosis assay and quantitative real-time polymerase chain reaction (PCR) in cervical cancer cell lines. The cell adhesion assay showed that staurosporine induces adhesion of cervical cancer cells to the extracellular matrix (ECM) protein fibronectin. The soft agar colony formation assay showed that staurosporine inhibits both the number and size of colony formation in a dose dependent manner and also induces adherent tendency in the cancer cells. Staurosporine also induces prominent apoptosis in single cell suspensions compared to adherent cells. Stroma cell induced transcription of matrix metalloprotease 1 (MMP1) and matrix metalloprotease 2 (MMP2) in cervical cancer cells was inhibited by staurosporine. Our results indicate that staurosporine induces anti-tumor response in the cervical tumor microenvironment by inhibiting the fundamental events for cancer progression and metastases. The present study represents an attractive area for further research and opens up new avenues towards the understanding of cervical cancer therapeutics.

CGMP inhibition of type 3 phosphodiesterase is the major mechanism by which C-type natriuretic peptide activates CFTR in the shark rectal gland

The in vitro perfused rectal gland of the dogfish shark (Squalus acanthias) and filter-grown monolayers of primary cultures of shark rectal gland (SRG) epithelial cells were used to analyze the signal transduction pathway by which C-type natriuretic peptide (CNP) stimulates chloride secretion. CNP binds to natriuretic receptors in the basolateral membrane, elevates cellular cGMP, and opens cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in the apical membrane. CNP-provoked chloride secretion was completely inhibitable by the nonspecific protein kinase inhibitor staurosporine and the PKA inhibitor H89 but insensitive to H8, an inhibitor of type I and II isoforms of cGMP-dependent protein kinase (cGKI and cGKII). CNP-induced secretion could not be mimicked by nonhydrolyzable cGMP analogs added alone or in combination with the protein kinase C activator phorbolester, arguing against a role for cGK or for cGMP-induced PKC signaling. We failed to detect a dogfish ortholog of cGKII by molecular cloning and affinity chromatography. However, inhibitors of the cGMP-inhibitable isoform of phosphodiesterase (PDE3) including milrinone, amrinone, and cilostamide but not inhibitors of other PDE isoenzymes mimicked the effect of CNP on chloride secretion in perfused glands and monolayers. CNP raised cGMP and cAMP levels in the SRG epithelial cells. This rise in cAMP as well as the CNP and amrinone-provoked chloride secretion, but not the rise in cGMP, was almost completely blocked by the Gαi-coupled adenylyl cyclase inhibitor somatostatin, arguing against a role for cGMP cross-activation of PKA in CNP action. These data provide molecular, functional, and pharmacological evidence for a CNP/cGMP/PDE3/cAMP/PKA signaling cascade coupled to CFTR in the SRG.

Drosophila Bestrophin-1 Currents Are Regulated by Phosphorylation via a CaMKII Dependent Mechanism

Cell swelling induced by hypo-osmotic stress results in activation of volume-regulated anion channels (VRAC) that drive a compensatory regulatory volume decrease. We have previously shown that the Best1 gene in Drosophila encodes a VRAC that is also activated by increases in intracellular Ca2+. The role of Best1 as a VRAC has recently been independently confirmed by the Clapham lab in an unbiased RNAi screen. Although dBest1 is clearly a volume-regulated channel, its mechanisms of regulation remain unknown. Here we investigate Drosophila Best1 (dBest1) regulation using the Drosophila S2 cell model system. Because dBest1 activates slowly after establishing whole-cell recording, we tested the hypothesis that the channel is activated by phosphorylation. Two experiments indicate that phosphorylation is required for dBest1 activation: nonspecific protein kinase inhibitors or intracellular perfusion with the non-hydrolyzable ATP analog AMP-PNP dramatically reduce the amplitude of dBest1 currents. Furthermore, intracellular perfusion with ATP-γ-S augments channel activation. The kinase responsible for dBest1 activation is likely Ca2+/calmodulin dependent kinase II (CaMKII), because specific inhibitors of this kinase dramatically inhibit dBest1 current activation. Neither specific PKA inhibitors nor inactive control inhibitors have effects on dBest1currents. Our results demonstrate that dBest1 currents are regulated by phosphorylation via a CaMKII dependent mechanism.

Ethanol Extract ofLepidium apetalumSeed Elicits Contractile Response and Attenuates Atrial Natriuretic Peptide Secretion in Beating Rabbit Atria

The seeds of Lepidium apetalum Willdenow (called “Tinglizi” in China and “Jungryukza” in Korea) have been used to discharge phlegm and improve dropsy in Oriental medicine. The present study investigated the effects of ethanol extract of the seeds of Lepidium apetalum (ELA) on atrial dynamics and atrial natriuretic peptide (ANP) secretion in beating rabbit atria. ELA increased atrial stroke volume, pulse pressure, and cAMP efflux, concomitantly attenuating ANP secretion in a dose-dependent manner. ELA-induced increases in atrial stroke volume, pulse pressure, and cAMP levels and decrease in ANP secretion were not inhibited by pretreatment with staurosporine, a nonspecific protein kinase inhibitor, or diltiazem and verapamil, the L-type Ca2+ channel blockers, respectively. Helveticoside, a well-known digitalis-like cardiac glycosidic constituent of ELA, also increased atrial dynamics, including stroke volume and pulse pressure, without changing cAMP efflux and ANP secretion, and the effects of helveticoside were not inhibited by pretreatment with staurosporine, diltiazem, and verapamil. These results suggest that the ELA-induced positive inotropic activity in beating rabbit atria might, at least partly, be due to the digitalis-like activity of helveticoside rather than an increase in cAMP efflux.

Inhibitor screening and selectivity assessment against multiple cellular protein kinases by capillary electrophoresis with laser-induced fluorescence detection

A method that can be used for screening protein kinase inhibitors (PKIs) and simultaneously assessing their selectivity is described. The method is based on simultaneously assaying multiple cellular protein kinases by performing capillary electrophoresis (CE) separation and measuring the peak areas of the phosphorylated substrate peptides. The powerful separation capability of CE combined with the highly sensitive and selective laser-induced fluorescence (LIF) detector enables the direct screening of PKIs against cell lysates, which are used as an inexpensive source of enzymes. Four cell lines, three specific substrate peptides labeled with 5-carboxyfluorescein (5-FAM), two relative specific PKIs (TBB and H-89) and one non-specific PKI (staurosporine) were utilized to prove the methodology. With this method, the inhibitory activity of the tested compounds against multiple protein kinases was identified in parallel by comparing the peak areas of the phosphorylated substrates with those obtained in the absence of any inhibitors. The reduced peak area of the phosphorylated substrate definitively represents a positive screening result. Simultaneously, assaying the inhibition of one inhibitor against mutiple cellular protein kinases enables the assessment of its selectivity. Compared to the conventional, single-target screening format, the cell lysate-based multi-target method is more informative, more straightforward and more cost effective.

Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells

Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H2O2 accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.

Oxytocin-induced elevation of ADP-ribosyl cyclase activity, cyclic ADP-riboseor Ca 2+ concentrations is involved in autoregulation of oxytocin secretionin the hypothalamus and posterior pituitary in male mice

Locally released oxytocin (OT) activates OT receptors (2.1:OXY:1:OT:) in neighboring neurons in the hypothalamus and their terminals in the posterior pituitary, resulting in further OT release, best known in autoregulation occurring during labor or milk ejection in reproductive females. OT also plays a critical role in social behavior of non-reproductive females and even in males in mammals from rodents to humans. Social behavior is disrupted when elevation of free intracellular Ca 2+ concentration ([Ca 2+] i) and OT secretion are reduced in male and female CD38 knockout mice. Therefore, it is interesting to investigate whether ADP-ribosyl cyclase-dependent signaling is involved in OT-induced OT release for social recognition in males, independent from female reproduction, and to determine its molecular mechanism. Here, we report that ADP-ribosyl cyclase activity was increased by OT in crude membrane preparations of the hypothalamus and posterior pituitary in male mice, and that OT elicited an increase in [Ca 2+] i in the isolated terminals over a period of 5 min. The increases in cyclase and [Ca 2+] i were partially inhibited by nonspecific protein kinase inhibitors and a protein kinase C specific inhibitor, calphostin C. Subsequently, OT-induced OT release was also inhibited by calphostin C to levels inhibited by vasotocin, an OT receptor antagonist, and 8-bromo-cADP-ribose. These results demonstrate that OT receptors are functionally coupled to membrane-bound ADP-ribosyl cyclase and/or CD38 and suggest that cADPR-mediated intracellular calcium signaling is involved in autoregulation of OT release, which is sensitive to protein kinase C, in the hypothalamus and neurohypophysis in male mice.

Pregnenolone sulfate modulation of N-methyl-d-aspartate receptors is phosphorylation dependent

Pregnenolone sulfate (PS), an endogenously occurring neurosteroid, has been shown to modulate the activity of several neurotransmitter-gated channels, including the N-methyl-d-aspartate receptor (NMDAR). NMDARs are glutamate-gated ion channels involved in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. To determine the mechanism that controls PS sensitivity of NMDARs, we measured NMDAR responses induced by exogenous agonist application in voltage-clamped HEK293 cells expressing NR1/NR2B NMDARs and cultured rat hippocampal neurons. We report that PS potentiates the amplitude of whole-cell recorded NMDAR responses in cultured hippocampal neurons and HEK293 cells; however, the potentiating effect of PS on NMDAR in outside-out patches isolated from cultured hippocampal neurons and HEK293 cells was lost within 2 min after patch isolation in a neurosteroid-specific manner. The rate of diminution of the PS potentiating effect was slowed by protein phosphatase inhibitors. Treatment of cultured hippocampal neurons with a nonspecific protein kinase inhibitor and a specific protein kinase A (PKA) inhibitor diminished PS-induced potentiation, which was recovered by adding a PKA, but not a protein kinase C (PKC), activator. These results suggest that the effect of PS on NMDARs is controlled by cellular mechanisms that are mediated by dephosphorylation/phosphorylation pathways.

Proteinase-activated Receptor 2 Stimulates Na,K-ATPase and Sodium Reabsorption in Native Kidney Epithelium

Proteinase-activated receptors 2 (PAR2) are expressed in kidney, but their function is mostly unknown. Since PAR2 control ion transport in several epithelia, we searched for an effect on sodium transport in the cortical thick ascending limb of Henle's loop, a nephron segment that avidly reabsorbs NaCl, and for its signaling. Activation of PAR2, by either trypsin or a specific agonist peptide, increased the maximal activity of Na,K-ATPase, its apparent affinity for sodium, the sodium permeability of the paracellular pathway, and the lumen-positive transepithelial voltage, featuring increased NaCl reabsorption. PAR2 activation induced calcium signaling and phosphorylation of ERK1,2. PAR2-induced stimulation of Na,K-ATPase Vmax was fully prevented by inhibition of phospholipase C, of changes in intracellular concentration of calcium, of classical protein kinases C, and of ERK1,2 phosphorylation. PAR2-induced increase in paracellular sodium permeability was mediated by the same signaling cascade. In contrast, increase in the apparent affinity of Na,K-ATPase for sodium, although dependent on phospholipase C, was independent of calcium signaling, was insensitive to inhibitors of classical protein kinases C and of ERK1,2 phosphorylation, but was fully prevented by the nonspecific protein kinase inhibitor staurosporine, as was the increase in transepithelial voltage. In conclusion, PAR2 increases sodium reabsorption in rat thick ascending limb of Henle's loop along both the transcellular and the paracellular pathway. PAR2 effects are mediated in part by a phospholipase C/protein kinase C/ERK1,2 cascade, which increases Na,K-ATPase maximal activity and the paracellular sodium permeability, and by a different phospholipase C-dependent, staurosporine-sensitive cascade that controls the sodium affinity of Na,K-ATPase.

CAMP PRODUCED BY PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE 27 INHIBITS ATRIAL NATRIURETIC PEPTIDE SECRETION IN RABBIT BEATING ATRIA

The aim of the present study was to determine the effects of increased cAMP levels in response to pituitary adenylate cyclase-activating polypeptide 27 (PACAP27) on atrial atrial natriuretic peptide (ANP) secretion in rabbit atria. A perfused beating atrial model was used in the present study and cAMP efflux and ANP levels in atrial perfusates were measured by radioimmnoassay. At 100 nmol/L, PACAP27 increased cAMP production, which resulted in subsequent inhibition of ANP secretion. Nicardipine (1.0 micromol/L), an L-type Ca2+ channel blocker, attenuated inhibition of ANP secretion by PACAP27. Staurosporine (1.0 micromol/L), a non-specific protein kinase inhibitor, and H-89 (1.0 micromol/L), a cAMP-dependent protein kinase A (PKA) inhibitor, completely blocked the inhibition of ANP secretion in response to PACAP27 but had no effect on PACAP27-induced increases in cAMP. In conclusion, the results suggest that increased cAMP levels in response to PACAP27 negatively regulate ANP secretion via the adenylate cyclase-cAMP-PKA signalling pathway in rabbit atria and that L-type Ca2+ channels may be involved, in part, in the regulation of ANP secretion by cAMP.

The positive inotropic effect of ethanol extract of Lepidium apetalum in beating rabbit atria

In the present study, the positive inotropic effects of ethanol extract of Lepidium apetalum (ELA) and possible mechanisms responsible for this effect were investigated in beating rabbit atria. ELA (5 x 10−4 g/ml) significantly increased atrial stroke volume, pulse pressure, and cAMP efflux in perfused beating rabbit atria. These effects were not altered by pre-treatment with diltiazem (5 x 10−6 M) or verapamil (1 x 10−6 M), L-type Ca2+ channel blockers. In addition, ELA-induced increases in atrial stroke volume, pulse pressure, and cAMP were not inhibited by pre-treatment of staurosporine (1 x 10−6 M), a non-specific protein kinase inhibitor. ELA markedly increased K+ concentration in beating atria-derived perfusate. Helveticoside (2 x 10−5 M), a well known digitalis-like cardiac glycosidic constituent of ELA, also increased atrial stroke volume and pulse pressure without changes in cAMP efflux, which were partially inhibited by pre-treatment with diltiazem, verapamil, or staurosporine, respectively. These results suggest that ELA-induced positive inotropic effect in beating rabbit atria might due to digitalis-like activity of helveticoside and partially be related with the increase in cAMP.

H-89, a Non-Specific Inhibitor of Protein Kinase A, Promotes Post-Ischemic Cardiac Contractile Recovery and Reduces Infarct Size

Myocardial ischemia is associated with increased production of cyclic adenosine monophosphate (cAMP), with potentially deleterious effects. We hypothesized that the ischemia-induced activation of cAMP-dependent protein kinase A (PKA), could beneficially be inhibited by a PKA-inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline-sulfonamide (H-89). H-89 when given to isolated perfused rat hearts before 30 minutes of global ischemia-reperfusion improved postischemic function and decreased infarct size. In another series, H-89 administered prior to preconditioning by 10 minutes of transient global ischemia decreased PKA activity (measured at the end of the preconditioning protocol) and augmented postischemic mechanical recovery. H-89 given for 5 minutes before the 10 minutes of transient ischemia further decreased infarct size from 13.4 +/- 1.0% (preconditioning alone) to 7.0 +/- 0.93 (P < 0.01). In a third series, forskolin (0.3 muM, 5 minutes, 10 minutes washout prior to ischemia) increased PKA activity and reduced infarct size. Prior H-89 decreased PKA activity after 5 minutes of forskolin and further reduced infarct size versus forskolin alone. In conclusion, three procedures increased postischemic recovery and reduced infarct size: H-89; preconditioning by transient ischemia; or forskolin as a preconditioning-mimetic. PKA-inhibition by H-89 further decreased infarct size beyond preconditioning or forskolin. Despite the reservation that H-89 could be non-selective in its actions, we propose H-89 as a candidate cardioprotective agent.

α-Tocotrienol provides the most potent neuroprotection among vitamin E analogs on cultured striatal neurons

Oxidative stress and apoptosis play pivotal roles in the pathogenesis of neurodegenerative diseases. We investigated the effects of vitamin E analogs on oxidative stress and apoptosis using primary neuronal cultures of rat striatum. A tocotrienol-rich fraction of edible oil derived from palm oil (Tocomin 50%), which contains α-tocopherol, and α-, γ- and δ-tocotrienols, significantly inhibited hydrogen peroxide (H 2O 2)-induced neuronal death. Each of the tocotrienols, purified from Tocomin 50% by high-performance liquid chromatography, significantly attenuated H 2O 2-induced neurotoxicity, whereas α-tocopherol did not. α-, γ- and δ-Tocotrienols also provided significant protection against the cytotoxicity of a superoxide donor, paraquat, and nitric oxide donors, S-nitrosocysteine and 3-morpholinosydnonimine. Moreover, tocotrienols blocked oxidative stress-mediated cell death with apoptotic DNA fragmentation caused by an inhibitor of glutathione synthesis, l-buthionine-[ S, R]-sulfoximine. In addition, α-tocotrienol, but not γ- or δ-tocotrienol, prevented oxidative stress-independent apoptotic cell death, DNA cleavage and nuclear morphological changes induced by a non-specific protein kinase inhibitor, staurosporine. These findings suggest that α-tocotrienol can exert anti-apoptotic neuroprotective action independently of its antioxidant property. Among the vitamin E analogs examined, α-tocotrienol exhibited the most potent neuroprotective actions in rat striatal cultures.

Biochemical characterization of the tobacco 42-kD protein kinase activated by osmotic stress.

In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family. The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA. A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed. Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase. The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases. Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK. A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity. In phosphorylation reactions, NtOSAK exhibited a preference for Mg(2+) over Mn(2+) ions and an inability to use GTP instead of ATP as a phosphate donor. The enzyme activity was not modulated by 5'-AMP. To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.

Characterization of dual specificity protein kinase from maize seedlings.

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.

Characterization of dual specificity protein kinase from maize seedlings.

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.