Myelin Basic Protein Kinase Research Articles

Annexin A6 mitigates neurological deficit in ischemia/reperfusion injury by promoting synaptic plasticity.

Alleviating neurological dysfunction caused by acute ischemic stroke (AIS) remains intractable. Given Annexin A6 (ANXA6)'s potential in promoting axon branching and repairing cell membranes, the study aimed to explore ANXA6's potential in alleviating AIS-induced neurological dysfunction. A mouse middle cerebral artery occlusion model was established. Brain and plasma ANXA6 levels were detected at different timepoints post ischemia/reperfusion (I/R). We overexpressed and down-regulated brain ANXA6 and evaluated infarction volume, neurological function, and synaptic plasticity-related proteins post I/R. Plasma ANXA6 levels were measured in patients with AIS and healthy controls, investigating ANXA6 expression's clinical significance. Brain ANXA6 levels initially decreased, gradually returning to normal post I/R; plasma ANXA6 levels showed an opposite trend. ANXA6 overexpression significantly decreased the modified neurological severity score (p = 0.0109) 1 day post I/R and the infarction area at 1 day (p = 0.0008) and 7 day (p = 0.0013) post I/R, and vice versa. ANXA6 positively influenced synaptic plasticity, upregulating synaptophysin (p = 0.006), myelin basic protein (p = 0.010), neuroligin (p = 0.078), and tropomyosin-related kinase B (p = 0.150). Plasma ANXA6 levels were higher in patients with AIS (1.969 [1.228-3.086]) compared to healthy controls (1.249 [0.757-2.226]) (p < 0.001), that served as an independent risk factor for poor AIS outcomes (2.120 [1.563-3.023], p < 0.001). This study is the first to suggest that ANXA6 enhances synaptic plasticity and protects against transient cerebral ischemia.

1,2-Dichloroethane induces cortex demyelination by depressing myelin basic protein via inhibiting aquaporin 4 in mice

1,2-Dichloroethane (1,2-DCE) is a pervasive environmental pollutant, and overexposure to this hazardous material causes brain edema and demyelination in humans. We found that 1,2-DCE inhibits aquaporin 4 (AQP4) and is a primary pathogenic effector of 1,2-DCE-induced brain edema in animals. However, AQP4 down-regulation’s link with cortex demyelination after 1,2-DCE exposure remains unclear. Thus, we exposed wild-type (WT) CD-1 mice and AQP4 knockout (AQP4-KO) mice to 0, 100, 350 and 700 mg/m3 1,2-DCE by inhalation for 28 days. We applied label-free proteomics and a cell co-culture system to elucidate the role of AQP4 inhibition in 1,2-DCE-induced demyelination. The results showed that 1,2-DCE down-regulated AQP4 in the WT mouse cortexes. Both 1,2-DCE exposure and AQP4 deletion induced neurotoxicity in mice, including increased brain water content, abnormal pathological vacuolations, and neurobehavioral damage. Tests for interaction of multiple regression analysis highlighted different effects of 1,2-DCE exposure level depending on the genotype, indicating the core role of AQP4 in regulation on 1,2-DCE-caused neurotoxicity. We used label-free quantitative proteomics to detect differentially expressed proteins associated with 1,2-DCE exposure and AQP4 inhibition, and identified down-regulation in myelin basic protein (MBP) and tyrosine-protein kinase Fyn (FYN) in a dose-dependent manner in WT mice but not in AQP4-KO mice. 1,2-DCE and AQP4 deletion separately resulted in demyelination, as detected by Luxol fast blue staining, and manifested as disordered nerve fibers and cavitation in the cortexes. Western blot and immunofluorescence confirmed the decreased AQP4 in the astrocytes and the down-regulated MBP in the oligodendrocytes by 1,2-DCE exposure and AQP4 inhibition, respectively. Finally, the co-culture results of SVG p12 and MO3.13 cells showed that 1,2-DCE-induced AQP4 down-regulation in the astrocytes was responsible for demyelination, by decreasing MBP in the oligodendrocytes. In conclusion, 1,2-DCE induced cortex demyelination by depressing MBP via AQP4 inhibition in the mice.

Electroacupuncture promotes regeneration and repair of myelin sheath of corpus callosum in demyelination mice

To explore the mechanism of electroacupuncture (EA) in accelerating the aggregation of microglia and promoting the remyelination at the location of demyelination. C57BL/6 mice were randomly divided into 4 groups: normal, control, model (LPC) and LPC＋EA. The demyelination model was established by microinjection of Lysolecithin (LPC, 1 µL) into the left corpus callosum. EA (2 Hz/15 Hz, 2－4 mA) was applied to "Baihui"(GV20)and "Zhiyang"(GV9)for 30 min，once daily for 3 days, then, once every other day for 18 days. Immuno-fluorescence staining was used to observe the expression of myelin basic protein (MBP) and Axl tyrosine kinase receptor (Axl), Iba1 and numbers of Olig2-positive oligodendrocytes in the corpus callosum. Western blot was employed to detect the expression of MBP in the corpus callosum, and Oil Red O staining was used to observe changes of number of myelin pieces. Following modeling, the expression levels of MBP on day 5 and 10 after modeling were significantly decreased (P<0.05, P<0.01), Iba1 expression and Olig2-positive oligodendrocyte numbers on day 10 apparently increased (P<0.001, P<0.01). On day 21 after modeling, the levels of the above mentioned indexes returned to normal. After EA intervention, the levels of MBP expression on day 5 and 10, Axl, Iba1 protein expression and Olig2-positive oligodendrocyte numbers on day 5 were markedly increased (P<0.001，P<0.01，P<0.05), while Iba1 expression on day 10 was considerably decreased in comparison with the model group (P<0.01)．Oil Red O staining showed that on day 5 after modeling, the number of red lipid droplets were obviously increased in the corpus callosum tissue on the injection side, and apparently reduced in the EA group, suggesting a clearance of the accumulated myelin fragments by EA. EA intervention may reduce myelin debris and promote the aggregation of microglial cells and oligodendrocytes to the injured site, accelerate the myelin regeneration and up-regulate the expression of MBP and Axl of corpus callosum in demyelination mice.

31 PROTOCOL OPTIMIZATION AND EVALUATION OF MATURATION PROMOTING FACTOR AND MITOGEN-ACTIVATED PROTEIN KINASE ACTIVITIES IN BOVINE CYTOPLASTS OBTAINED BY CHEMICAL ENUCLEATION TECHNIQUES

Chemical enucleation using microtubule-depolymerizing drugs is an attractive procedure to simplify the enucleation process in nuclear transfer. The aim of this study was to optimize chemically assisted (CA) and chemically induced (CI) enucleation protocols using metaphase II (MII) and pre-activated bovine oocytes, respectively, and to evaluate the activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) in cytoplasts generated by these techniques. Initially, we determined the shortest effective treatment of MII and activated oocytes with 0.05 μg mL–1 demecolcine. Bovine oocytes in vitro matured (IVM) for 19 h (MII) or activated artificially with 5 μM ionomycin (5 min) and 10 μg mL–1 cycloheximide (5 h) after 26 h IVM were treated with demecolcine and samples were collected at 0, 0.25, 0.5, 1.0, 1.5, and 2.0 h of treatment. Oocytes were then stained with 10 μg mL–1 Hoechst 33342 and the protrusion or enucleation rates were determined. Next, we evaluated histone H1 and myelin basic protein (MBP) kinases, reflecting MPF and MAPK activities, respectively, in oocytes obtained from these treatments, and for that we used the method described by Kubelka et al. (2000 Biol. Reprod. 62, 292–302). Protrusion and enucleation rates were evaluated by the chi-squared (χ2) test, and MPF and MAPK activities were submitted to ANOVA and Tukey's test at 5% significance. For MII oocytes, effects of demecolcine were observed as early as 15 min, with a significant difference (P &lt; 0.05) between control (12/112, 10.7%) and treated (33/114, 28.9%) groups in relation to protrusion rates. The largest number of protrusions was observed after 1.0 h of treatment (control: 15/113, 13.3%a; treated: 45/111, 40.5%b). In pre-activated oocytes, effects of demecolcine were also observed after 15 min, and in both techniques there were no significant differences between groups treated with demecolcine for 1.0, 1.5, or 2.0 h (CA: 40.5 to 52.5% of protrusion; CI: 35.2 to 46.7% of enucleation). In contrast to previous reports in which high concentrations of demecolcine for CA enucleation increased MPF activity, we observed no alterations in the activity of this factor at a demecolcine concentration of 0.05 μg mL–1. Activity of MAPK also did not differ significantly between the control and treated groups throughout evaluation. In the CI technique, a significant difference in MPF activity was observed after 0.5 h (70.3%) and 2.0 h of activation (39.1%), considering that the activity was 100% at the beginning of the evaluation. However, we observed no significant difference between the control and treated groups at any of the time points studied, as verified for MAPK activity. The exact effect of MPF on the nucleus in mammals is not well established. We believe that the use of low concentrations of demecolcine for short periods is less damaging to embryonic development and, until we have a better understanding of the effect of these kinases on the transferred nucleus, we recommend its use for chemical enucleation protocols in bovine. Financial support: FAPESP 2010/20744-6 and 2011/12983-3.

Biochemical identification of the OsMKK6–OsMPK3 signalling pathway for chilling stress tolerance in rice

MAPK (mitogen-activated protein kinase) pathways have been implicated in stress signalling in plants. In the present study, we performed yeast two-hybrid screening to identify partner MAPKs for OsMKK (Oryza sativa MAPK kinase) 6, a rice MAPK kinase, and revealed specific interactions of OsMKK6 with OsMPK3 and OsMPK6. OsMPK3 and OsMPK6 each co-immunoprecipitated OsMKK6, and both were directly phosphorylated by OsMKK6 in vitro. An MBP (myelin basic protein) kinase assay of the immunoprecipitation complex indicated that OsMPK3 and OsMPK6 were activated in response to a moderately low temperature (12°C), but not a severely low temperature (4°C) in rice seedlings. A constitutively active form of OsMKK6, OsMKK6DD, showed elevated phosphorylation activity against OsMPK3 and OsMPK6 in vitro. OsMPK3, but not OsMPK6, was constitutively activated in transgenic plants overexpressing OsMKK6DD, indicating that OsMPK3 is an in vivo target of OsMKK6. Enhanced chilling tolerance was observed in the transgenic plants overexpressing OsMKK6DD. Taken together, our data suggest that OsMKK6 and OsMPK3 constitute a moderately low-temperature signalling pathway and regulate cold stress tolerance in rice.

Okadaic acid-sensitive phosphatase is related to MII/G1 transition in mouse oocytes

It is reported that okadaic acid (OA)-sensitive phosphatase is related to mitogen-activated protein kinase (MAPK)/p90rsk activation in mammalian oocytes. OA is also involved in the positive feedback loop between M phase-promoting factor (MPF) and cdc25c in Xenopus oocytes during meiotic maturation. However, the effect of phosphatase inhibition by OA on MPF and MAPK activities at the MII/G1 in oocytes remains unknown. The aim of this study is to clarify the relationship between OA-sensitive phosphatase and mitosis MII/G1 transition in mouse oocytes. MII-arrested oocytes were, isolated from mice, inseminated and cultured in TYH medium (control group) or TYH medium supplemented with 2.5 μM of OA (OA group). Histone H1 kinase and myelin basic protein (MBP) kinase activities were measured as indicators of MPF and p42 MAPK activities after insemination. Phosphorylation of cdc25c after insemination was analized in OA and control group by western blotting. Seven hours after insemination a pronucleus (PN) was formed in 84.1% (69/85) of oocytes in the control group. However, no PN was formed in oocytes of the OA group (p < 0.001). Although MPF and MAPK activities in the control group significantly decreased at 3, 4, 5, and 7 h after insemination, these decreases were significantly inhibited by OA addition (p < 0.05). Furthermore, OA addition prevented cdc25c dephosphorylation 7 h after insemination. In conclusion, OA-sensitive phosphatase correlates with inactivation of MPF and MAPK, and with the dephosphorylation of cdc25c at the MII/G1 transition in mouse oocytes.

Hexavalent chromium (VI) stress induces mitogen-activated protein kinase activation mediated by distinct signal molecules in roots of Zea mays L.

Hexavalent chromium (VI) is a cytotoxic metal ion in plants. However, the mechanisms involved in the cellular response to the metal have not yet been well established. In plants, mitogen-activated protein kinase (MAPK) cascades play an important role in signal transduction related to biotic and abiotic stresses. In the present study, we investigated the Cr(VI)-induced MAPKs activation and the correlative mechanism of activation in maize ( Zea mays L.) roots. Cr(VI) elicited a remarkable increase in a 45-kDa myelin basic protein (MBP) kinase activity with MAPK-like characteristics, which was identified as ZmMPK5 by immunokinase and immunoblot assays. Pretreatment with DMTU, a peroxide hydrogen (H 2O 2) scavenger, and DPI, a NADPH oxidase inhibitor, the Cr(VI)-induced ZmMPK5 activation was almost completely suppressed, suggesting that Cr(VI)-activated ZmMPK5 requires for H 2O 2. Application of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, could activate ZmMPK5. Pretreatment with cPTIO and l-NAME, a NO scavenger and a nitric oxide synthase (NOS) inhibitor, respectively, Cr(VI)-induced ZmMPK5 activation was attenuated effectively, implying that NO is involved in Cr(VI)-activated ZmMPK5. Furthermore, a calcium-dependent protein kinase (CDPK) antagonist, W7, abolished Cr(VI)-stimulated ZmMPK5 activation, indicating that CDPKs may participate in the ZmMPK5 activation. The results obtained suggest that Cr(VI)-induced activation of ZmMPK5, a candidate for MAPK signaling cascades, can be modulated by other distinct signaling pathways.

Cerebrospinal fluid levels of myelin basic protein and creatin kinase BB as index of active demyelination

Radioimmunoassay-determined myelin basic protein (MBP) shed to CSF during active demyelination, has been found to be a useful but non-specific test for MS. CSF creatin kinase BB (CK-BB), as measured by radioimmunoassay, is increased in a variety of neurological diseases, and has been considered a useful indication of brain damage but not of demyelinating diseases. Taking into account that the mean concentration of CSF CK-BB should not be increased in patients during the acute phase of MS, we suggest that the CSF MBP/CK-BB ratio could be a more specific index to demyelination than CSF-MBP alone. We also defined a laboratory demyelination pattern (CSF MBP greater than mean control MBP + 2 S.D. and CK-BB less than MBP). CSF levels of MBP and CSF levels of CK-BB were determined by radioimmunoassay in 232 patients with several neurological disorders and 33 control subjects. Patients diagnosed as having MS with clinical exacerbation had significantly higher values of CSF-MBP/CSF-BB ratio than control subjects. Our study showed a significant presence of demyelination pattern in CSF of patients with MS. We conclude that the CSF MBP/CK-BB ratio and the CSF demyelination pattern may be new and reliable tests for the diagnosis of MS.

ROS and CDPK-like kinase-mediated activation of MAP kinase in rice roots exposed to lead

Lead (Pb2+) is a cytotoxic metal ion in plants, the mechanism of which is not yet established. The aim of this study is to investigate the signalling pathways that are activated by elevated concentrations of Pb2+ in rice roots. Root growth was stunted and cell death was accelerated when exposed to different dosages of Pb2+ during extended time periods. Using ROS-sensitive dye and Ca2+ indicator, we demonstrated that Pb2+ induced ROS production and Ca2+ accumulation, respectively. In addition, Pb2+ elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot and immunoprecipitation analysis, 40- and 42-kDa MBP kinases that were activated by Pb2+ were identified to be mitogen-activated protein (MAP) kinases. Pre-treatment of rice roots with an antioxidant and a NADPH oxidase inhibitor, glutathione (GSH) and diphenylene iodonium (DPI), effectively reduced Pb2+-induced cell death and MAP kinase activation. Moreover, calcium-dependent protein kinase (CDPK) antagonist, W7, attenuated Pb2+-induced cell death and MAP kinase activation. These results suggested that the ROS and CDPK may function in the Pb2+-triggered cell death and MAP kinase signalling pathway in rice roots.

UVB/UVA Radiation Activates a 48 kDa Myelin Basic Protein Kinase and Potentiates Wound Signaling in Tomato Leaves

We investigated the effect of UV radiation on early signaling events in the response of young tomato plants (Lycopersicon esculentum) to wounding. Ultraviolet-C (< 280 nm) and UVB/UVA (280-390 nm) radiation both induced 48 kDa myelin basic protein kinase activity in leaves. The activation was associated with phosphorylation of tyrosine residues on the kinase, which is indicative of protein kinases of the mitogen-activated protein kinase family. Ultraviolet-C irradiation resulted in a strong proteinase inhibitor synthesis, as reported previously (Conconi et al., Nature 383, 826-829, 1996). Under the conditions used, UVB/UVA radiation did not induce proteinase inhibitor synthesis but resulted in a strong potentiation of systemic proteinase inhibitor synthesis in response to wounding. The UVB/UVA-irradiated plants that were subsequently wounded accumulated 2.5-4-fold higher levels of proteinase inhibitor I when compared to wounded non-irradiated plants. The potentiating effect was most prominent in the systemic unwounded leaf of a wounded plant. Levels of 12-oxo-phytodienoic acid and jasmonic acid that have been well documented to increase in response to wounding were not detected in response to UVB/UVA irradiation alone. The effect of UVB/UVA radiation in potentiating plant defense signaling should be further considered as a factor that may influence the ecological balance between plants and their predators.

Early events in the signalling pathway for the activation of MAPKs in rice roots exposed to nickel.

It is well known that small quantities of nickel (Ni) are essential for plant species, and higher concentrations of Ni retard plant growth. However, the molecular mechanisms responsible for the regulation of plant growth by Ni are not well understood. The aim of this study is to investigate the early signalling pathways activated by Ni on rice (Oryza sativa L.) root. We showed that Ni elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot and immunoprecipitation analyses, it is suggested that Ni-activated 40- and 42-kDa MBP kinases are mitogen-activated protein kinases (MAPKs). Pretreatment of rice roots with the antioxidant, glutathione (GSH), the phospholipase D (PLD) inhibitor, n-butanol, and the calmodulin and CDPK antagonist and W7 inhibited Ni-induced MAPK activation. These results suggest that various signalling components are involved in transduction of the Ni signal in rice roots.

Distinct signalling pathways for induction of MAP kinase activities by cadmium and copper in rice roots

Plant growth is severely affected by toxic concentrations of heavy metals. On characterizing the heavy metal-induced signalling pathways, the effects of cadmium (CdCl2) and copper (CuCl2) on MBP (myelin basic protein) kinase activities in Oryza sativa L. cv. TNG67 were analysed and it was found that Cd2+-induced 42 kDa MBP kinase has the characteristics of a mitogen-activated protein (MAP) kinase. This study confirmed that the 42 kDa kinase-active band contains, at least, the activities of OsMPK3 and OsMPK6. Then, the heavy metal signal transduction pathways leading to MAP kinase activation in rice roots were examined. Pretreatment with sodium benzoate, a hydroxyl radical scavenger, attenuated Cd2+- or Cu2+-induced MAP kinase activation. The Cd2+-, but not Cu2+-, induced MAP kinase activities were suppressed by diphenylene iodonium (DPI), an NADPH oxidase inhibitor, and Cd2+ induced NADPH oxidase-like activities, suggesting that NADPH oxidases may be involved in Cd2+-induced MAP kinase activation. Using a Ca2+ indicator, it was demonstrated that Cd2+ and Cu2+ induce Ca2+ accumulation in rice roots. The Cd2+- and Cu2+-induced MAP kinase activation required the involvement of Ca2+-dependent protein kinase (CDPK) and phosphatidylinositol 3-kinase (PI3 kinase) as shown by the inhibitory effect of a CDPK antagonist, W7, and a PI3 kinase inhibitor, wortmannin, respectively. Furthermore, bongkrekic acid (BK), a mitochondrial permeability transition pore opening blocker, suppressed Cd2+-, but not Cu2+-, induced MAP kinase activation, indicating that Cd2+-induced MAP kinase activities are dependent on the functional state of mitochondria. Collectively, these findings imply that Cd2+ and Cu2+ may induce MAP kinase activation through distinct signalling pathways. Moreover, it was found that the 42 kDa MAP kinase activities are higher in Cd-tolerant cultivars than in Cd-sensitive cultivars. Therefore, the Cd-induced 42 kDa MAP kinase activation may confer Cd tolerance in rice plants.

Peptide Microarray Analysis of Substrate Specificity of the Transmembrane Ser/Thr Kinase KPI-2 Reveals Reactivity with Cystic Fibrosis Transmembrane Conductance Regulator and Phosphorylase

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Effects of iron excess on cell viability and mitogen‐activated protein kinase activation in rice roots

Iron is an essential micronutrient for normal growth and development of plants. However, at high concentrations, iron can become toxic to plants. Very little information is known about the molecular mechanism responsible for the regulation of plant growth by excess iron. The aim of this study was to investigate the signal transduction pathway activated by increasing concentrations (0.5, 1.0, and 2.5 mm) of iron. We showed that iron elicited a remarkable myelin basic protein (MBP) kinase activity. By Western blot and immunoprecipitation analysis, we suggested that iron‐activated 42‐kDa MBP kinase is a mitogen‐activated protein kinase (MAPK). Cell death in rice roots due to iron toxicity was investigated using inhibitors of signal molecules known to regulate programmed cell death in plants. Phenylarsine oxide (PAO) and sodium orthovanadate, known inhibitors of tyrosine phosphatase, reduced iron‐induced root cell death, but cantharidin and endothall two‐serine/threonine phosphatase inhibitor enhanced iron‐induced root cell death. Moreover, our results revealed that H+‐ATPase might participate in iron‐induced cell death. These results suggested that the MAPK, reactive oxygen species, protein phosphatase, and H+‐ATPase might function in the plant iron‐triggered signaling pathway in rice roots.

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.

A Family Portrait of the RIO Kinases

The RIO family of atypical serine protein kinases has been first characterized only recently. It consists of enzymes that contain a unique domain with a characteristic kinase sequence motif and usually some additional domains. At least two RIO proteins, Rio1 and Rio2, are present in organisms varying from Archaea to humans, with a third Rio3 subfamily present only in multicellular eukaryotes. Yeast Rio1 and Rio2 proteins have been implicated in the processing of 20 S pre-rRNA and are necessary for survival of the cells. Crystal structures of Archaeoglobus fulgidus Rio1 and Rio2 have shown that whereas the overall fold of these enzymes resembles typical protein kinases, some of the structural domains, particularly those involved in peptide substrate binding, are not present. The mode of binding of nucleotides also differs from that found in typical protein kinases. Although it has been shown that both Rio1 and Rio2 have the enzymatic activity of kinases and are capable of autophosphorylation, the biological substrates of RIO proteins and their full biological role still remain to be discovered.

Zinc induces mitogen-activated protein kinase activation mediated by reactive oxygen species in rice roots

It is well known that zinc (Zn) is one of the micronutrients essential for normal growth and development of plants. However, the molecular mechanisms responsible for the regulation of plant growth by Zn are still not completely understood. The aim of this study was to investigate the signalling transduction pathways activated by Zn. We show that Zn elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot analysis, we suggest that Zn-activated 40- and 42-kDa MBP kinases are mitogen-activated protein kinases (MAPK). Pre-treatment of rice roots with reactive oxygen species (ROS) scavenger, sodium benzoate, was able to effectively prevent Zn-induced MAPK activation. However, phosphoinositide 3-kinase (PI-3K) inhibitor, LY294002, was unable to inhibit Zn-induced MAPK activation. These results suggest that the ROS may function in the Zn-triggered MAPK signalling pathway in rice roots.

Activation of stress-responsive mitogen-activated protein kinase pathways in hybrid poplar (Populus trichocarpa x Populus deltoides)

Plant mitogen-activated protein kinase (MAPK) cascades are important amplifying modules that can rapidly transduce stress signals into various appropriate intracellular responses. Several extracellular regulated kinase (ERK)-type MAPKs involved in plant defense signaling have been identified in herbaceous species, but no MAPK cascade has yet been characterized in a tree species. We examined the signal transduction events that lead to activation of defense mechanisms in poplar, a major forest species of economic and ecological importance which is becoming the model tree system for studying stress and adaptation responses. We show that, in poplar cell suspensions and leaf tissue, chitosan, a non-host-specific elicitor, and ozone, a strong oxidant and atmospheric pollutant, induce rapid and transient activation of at least two myelin basic protein (MBP) kinases with apparent molecular masses of 44 and 47 kD. The chitosan- and ozone-activated kinases have characteristics of MAPKs-they preferentially phosphorylate MBP, require tyrosine and threonine phosphorylation to be activated and are specifically recognized by anti-ERK and anti-pERK antibodies. Moreover, activation of these poplar MAPKs by chitosan or ozone is dependent on the production of reactive oxygen species; the influx of calcium ions via membrane channels; the activation of an upstream, membrane-localized component; and a cognate MAPK kinase (MAPKK). These data suggest that biotic and abiotic challenges activate MAPKs in poplar, as in herbaceous species, which then function as a convergence point for pathogen defense and oxidant stress signaling cascades.

Abscisic acid promoted changes in the protein profiles of rice seedling by proteome analysis

This study investigates the influence of exogenously applied abscisic acid (ABA) on the leaves and leaf sheaths of two-week-old rice seedling at the level of the proteome. Significant differences were observed in the two-dimensional polyacrylamide gel electrophoresis protein profiles between control and ABA treated samples. Amino-acid sequence analysis of affected proteins revealed that ABA caused drastic changes in the major photosynthetic protein, ribulose 1,5-bisphosphate carboxylase/oxygenase and accumulation of certain defense/stress-related proteins. Moreover, cutting or treating leaf sheaths with jasmonic acid (JA) rapidly increased the endogenous level of ABA, suggesting a role for ABA during the defense/stress-response. Comparative study indicated a potential overlap between ABA and JA as detected at the level of the proteome. Furthermore, in vitro protein phosphorylation experiments and in-gel kinase assays also revealed considerable changes in the phosphorylation status of some proteins, and differential effects on myelin basic protein and calcium-dependent protein kinase activities by ABA treatment, which suggests involvement of kinase in the downstream signaling cascade. These results provide evidence at proteome level for the involvement of ABA in stress-response in rice seedling.

Activation of a 36-kDa Myelin Basic Protein Kinase during Cadmium-Induced Apoptosis in Human Leukemia HL-60 Cells

Cadmium (Cd) is a well-known modulator of intracellular signal transduction including mitogen-activated protein kinases and protein kinase C. In this study, we investigated activation of a kinase using myelin basic protein (MBP) as a substrate during Cd-induced apoptosis in human leukemia HL-60 cells. To detect a kinase during Cd-induced apoptosis in HL-60 cells, we performed an in-gel kinase assay using MBP as a substrate and found that Cd induced the activation of a kinase with an apparent molecular mass of 36 kDa. The time-course of appearance of DNA ladders induced by Cd was consistent with that of activation of this kinase. The kinetics of activation of p36 MBP kinase was different from that of p38 mitogen-activated protein kinase (p38MAPK). Activation of p36 MBP kinase was also observed with kinetics distinct from that of activation of p38MAPK during mercuric chloride-induced apoptosis. This is the first report on activation of p36 MBP kinase during Cd- or Hg-induced apoptosis.